Fig. 6

RBX1 regulates PKM alternative splicing via SMAR1/HDAC6 complex degradation. A Co-IP showing direct binding of endogenous RBX1 and SMAR1 in CAL62 cells. B GST pull-down assay showing direct binding of endogenous RBX1 and SMAR1. C Top-ranked docking confirmation. 3D structures of SMAR1 and RBX1, with SMAR1 and RBX1 shown in green and cyan. D Western blotting was performed to detect the expression of RBX1 and SMAR1 in different groups. E, F qRT-PCR was performed to detect the expression of RBX1 and SMAR1 in different groups. G. CAL62 cells were transfected with shRBX1 plasmid. After that, cells were exposed to 20 μmol/L cyclohexanone (CHX) at the given times, and the SMAR1 degradation was identified using western blotting. H 8305C cells were treated with 10 μM MG132, while the RBX1 expression was altered. The SMAR1 protein expression level was determined by western blotting. I CAL62 cells were treated with 10 μM MG132 while transfecting them with HA-RBX1 or shRBX1 plasmids. Subsequently, the level of ubiquitin bound to the SMAR1 protein was measured by Co-IP. J Wild-type SMAR1 or K- to -R mutations in ATC cells (mutations in all the Lys positions of SMAR1 gene) for ubiquitination. K Determination of the type of SMAR1 ubiquitination in ATC cells. L Western blotting showing expression levels of RBX1, SMAR1, HDAC6, and PKM2 in shRBX1-CAL62 cells. M Co-IP combined with western blotting showing the expression levels of SMAR1 and HDAC6 in shRBX1-CAL62 cells. N Western blotting showing expression levels of RBX1, SMAR1, HDAC6, and PKM2 in HA-RBX1-8305C cells. O Co-IP combined with western blotting showing the expression levels of SMAR1 and HDAC6 in HA-RBX1-8305C cells